Alcohol and congenital heart defects: an experimental study in mice.

Pregnant mice were exposed to a single dose of alcohol (0.03 ml of 25% alcohol X g-1 mouse) or two doses (2 X 0.015 ml of 25% alcohol X g-1 mouse) 4 h apart, by intraperitoneal injection or by gavage, on days 7, 8, 9 or 10 of gestation. The mice were killed on the day before term and the fetuses examined for heart defects. Alcohol exposure on day 8, 9 or 10 of gestation caused a high incidence of ventricular septal defects (60%, 75% and 15% respectively). Defects of both the membranous and muscular parts of the septum were seen as well as more complex ventricular septal defects involving the great vessels. Day 7 was resistant to the induction of heart defects. This study has demonstrated that a relatively short exposure to high doses of alcohol during pregnancy in mice can cause congenital heart defects. This has important implications both as a possible cause of congenital heart anomalies in humans and for the counselling of mothers of affected children.

A review of the contribution of whole embryo culture to the determination of hazard and risk in teratogenicity testing.

Whole embryo culture appears to be an excellent method to screen chemicals for teratogenic hazard. Compared to in vivo testing it is cheap and rapid and does not involve experimentation on live adult animals. Also in the important area of risk estimation whole embryo culture offers distinct advantages over in vivo teratogenicity testing. Adverse embryonic outcomes (malformations or embryotoxicity) are directly related to the serum concentration of the compound being tested and can be compared to the serum concentration in the human. A similar comparison is not possible after in vivo testing because for most compounds there are major pharmacokinetic differences between humans and experimental animals. In vivo testing is also limited by the possibility that metabolites that occur in the human do not occur in the test animal. This problem can be overcome in the in vitro system by adding the metabolite directly at the desired concentration either with or without the parent compound. There is only one major disadvantage to in vitro testing and that is the limited period of embryogenesis that is undertaken in the commonly used culture system. This restricts the range of malformations that can be induced and may render the testing system unsuitable for compounds that are likely to exert their major toxicological effect late in gestation. Any evaluation of whole embryo culture for hazard and risk assessment in teratology must take into account the limited value of currently used in vivo methods. Over 2000 chemicals have been reported to be teratogenic in experimental animals exposed in vivo (Shepard, Catalog of Teratogenic Agents, 1989). In comparison only about 20 chemicals are known to cause birth defects in the human. This large number of in vivo false-positive cannot easily be distinguished from true-positives. In this respect in vivo testing is severely deficient. The embryo culture testing system would also be expected to produce many false-positives; but by comparing effective drug concentrations with human therapeutic concentrations they can be differentiated from true-positives. The most serious deficiency for an in vivo or in vitro teratogenicity testing system would be false-negatives. This has not been a problem in the validation of in vitro testing so far (except perhaps procarbazine), but difficult drugs such as thalidomide were not included. Thalidomide remains an important index chemical because it is not teratogenic in rats or mice but is teratogenic in the rabbit and human. It is likely that these species differences are due to metabolic differences between species and it is possible that if the proximate teratogen/s of thalidomide were identified they would be teratogenic in rat embryo culture. Whole embryo culture remains a very powerful technique that should continue to contribute to the determination of the safety of drugs and other chemicals during pregnancy.

The toxic effects of cadmium on the neonatal mouse CNS.

Mice (QS outbred strain) received a single subcutaneous injection of cadmium chloride in saline on postnatal day 1, 8, 15, or 22. Histological examination, 24 hours after cadmium exposure on day 1, revealed petechial hemorrhages, edema, and cellular pycnosis throughout much of the immature brain. Treatment on days 8 or 15 produced similar damage, particularly edema and pycnosis, but affected progressively less of the brain until, by day 22, the brain was apparently unaffected by cadmium. Some animals, allowed to survive six to eight weeks after cadmium injection, showed behavioral anomalies and persistent brain deficits. Electron microscopic examination of parietal cortex from animals exposed to cadmium on day 1 revealed that petechial hemorrhages first occurred tow hours after treatment. The hemorrhages increased during the next six hours, and were accompanied by thinning and vacuolization of the capillary walls and widening of interendothelial gaps. In general, such changes were restricted to the partially differentiated capillaries. Degenerative changes in the brain cells were first seen about six hours after cadmium exposure.

Interference with gastrulation during the third week of pregnancy as a cause of some facial abnormalities and CNS defects.

During the third week of pregnancy the human embryo undergoes a major developmental process, gastrulation, during which the two-layered embryo is converted into a three-layered embryo. At the same time, the upper epiblast layer is induced to form the neural plate. Evidence is presented which suggests that interference with this process by genetic, physical, or chemical agents can cause a range of CNS abnormalities and facial abnormalities, including those described as characteristic of the FAS.

Severe cervical dysplasia and nasal cartilage calcification following prenatal warfarin exposure.

We present an infant who was exposed to warfarin throughout pregnancy and has warfarin embryopathy. When the child was examined radiologically at 20 months areas of calcification were visible in the septal and alar cartilages of the small external part of the nose. The location of this ectopic calcification is consistent with that seen in an animal model of the warfarin embryopathy. It supports the hypothesis that warfarin interferes with the prenatal growth of the cartilaginous nasal septum by inhibiting the normal formation of a vitamin K-dependent protein that prevents calcification of cartilage. The child also had severe abnormalities of the cervical vertebrae and secondary damage to the spinal cord. Cervical vertebral anomalies are a relatively common finding in the warfarin embryopathy and in the related Binder syndrome.

Prenatal exposure to phenytoin, facial development, and a possible role for vitamin K.

Ten patients with maxillonasal hypoplasia (Binder "syndrome"), who were prenatally exposed to phenytoin (usually in combination with other anticonvulsants), were identified retrospectively. In addition to their facial anomalies, 6 of the patients were radiographed neonatally and showed punctate calcification, characteristic of chondrodysplasia punctata. Evidence is presented that the facial abnormalities seen in these children are due to anticonvulsant-induced vitamin K deficiency, causing abnormal development of the cartilaginous nasal septum. We propose that early vitamin K supplementation of at-risk pregnancies may prevent the development of maxillonasal hypoplasia, which in some patients is severely disfiguring and causes great emotional distress. Correction of this facial defect requires surgical and dental treatment over a long period of time.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

Increased vascularity and alterations in cytochrome oxidase distribution associated with abnormal hippocampal cytoarchitecture in micrencephalic rats.

Prenatal exposure of the developing rat brain to methylazoxymethanol acetate results in the formation of ectopic groups of pyramidal neurons in the subfields CA1 and CA2 of the mature hippocampal formation. These ectopic neurons are situated in regions of increased vascularization and increased activity of cytochrome oxidase, as demonstrated histochemically. Both these findings suggest that there may be an abnormality of distribution or intensity of oxidative metabolic activity in the ectopic pyramidal neurons found in these animals.

Vascularity and cytochrome oxidase distribution in the occipital cortex in MAM Ac-induced micrencephaly.

The laminar distributions of cytochrome oxidase activity and vascularization of the occipital cortex of animals made micrencephalic by exposure to MAM Ac on E14 or E16 were examined and compared to control animals. Despite severe disruptions of the normal cytoarchitecture in MAM Ac exposed animals, the laminar distributions of vascular profiles, branch points and cytochrome oxidase activity in micrencephalic animals were similar to those in control animals.

A new mouse mutant showing cerebellar abnormalities.

This paper describes a previously unreported recessive mutation in mice which, in the homozygous state, caused an abnormal posture, characterised by extension and abduction of the limbs on one side. This position alternated causing the mice to fall to one side or the other. The only histological abnormality noted, in the slightly smaller brains of the mutants, was a displacement and disarrangement of the Purkinje cells into the granular layer, in parts of the nodulus.

The choroid plexus as a target site for cadmium toxicity following chronic exposure in the adult mouse: an ultrastructural study.

The effects on the choroid plexus (CP) of chronic (22 weeks) exposure to 1, 10 or 100 ppm Cd2+ in the drinking water was studied in adult mice. The CP from these mice was examined using scanning and transmission electron microscopy. The mice receiving 10 or 100 ppm Cd2+ showed a dose dependent reduction in fluid intake and weight loss. The CP from animals receiving 100 ppm Cd2+ showed severe degenerative changes characterized by loss of microvilli, rupturing of the apical surface, increased cytoplasmic vacuolation and cellular debris. There was also an increase in cellular blebbing and increased dark/light cell ratio. Cerebrospinal fluid analysis revealed an increased protein content. The mice receiving 10 ppm Cd2+ showed similar but less severe degenerative changes. The 1 ppm Cd2+ group were indistinguishable from controls. These results suggest that it would be appropriate to examine CP function in individuals excessively exposed to Cd.

Retention and distribution of manganese in the mouse brain following acute exposure on postnatal day 0, 7, 14 or 42: an autoradiographic and gamma counting study.

The relationship between postnatal age at time of Mn exposure, and the retention and distribution of Mn in the brain of the mouse was studied using gamma counting and autoradiography. A single intraperitoneal dose of Mn2+ (11 micrograms/kg or 25 mg/kg) containing radioactive 54Mn was administered on postnatal day 0, 7, 14 or 42. Animals were then killed at various times post exposure (1-114 days) and retention and distribution determined. Maximum retention of Mn in the brain (calculated as a percentage of administered dose) occurred 43 days following exposure on day 0. For day 7, 14 or 42 the highest Mn retention was 24 h after exposure. Maximum retention was 2.9%, 3.5%, 2.5% or 0.3% respectively, Mn retention after 114 days was 0.4%, 0.3%, 0.3% or 0.04% respectively. For the carrier-added group maximum retention was lower with corresponding figures of 2.5%, 2.4%, 1.1% or 0.2% and retention after 114 days was also lower 0.1%, 0.06%, 0.05% or 0.01%. The autoradiography studies indicated that Mn was located in areas of the brain which had a high perikaryal density at the time of exposure and in the choroid plexus. High and low doses of Mn were similarly distributed and there was little change in distribution with time.

The choroid plexus and cerebral vasculature as target sites for cadmium following acute exposure in neonatal and adult mice: an autoradiographic and gamma counting study.

The relationship between postnatal age at time of cadmium (Cd) exposure, and the retention and distribution of Cd in the central nervous system (CNS) was studied using gamma counting and autoradiography. A single intraperitoneal dose of Cd2+ (84 micrograms/kg or 750 micrograms/kg) containing radioactive 109Cd, was administered on postnatal day 0, 7, 14, or 42. Animals were then killed at various times post exposure (1-115 days) and retention and distribution determined. Maximum retention of Cd in the brain (calculated as a percentage of administered dose) occurred 1-24 days after exposure. Following exposure on day 0, 7, 14, or 42, maximum retention was 1.2%, 1.6%, 0.4%, or 0.09%, respectively. Cd retention after 114 days was 0.3%, 1.0%, 0.1%, or 0.04% for the respective days of exposure. For the carrier-added group maximum retention was higher with corresponding figures of 2.3%, 1.6%, 1.4%, or 0.07% and retention after 114 days was also elevated 0.6%, 1.1%, 0.4%, or 0.04%. The autoradiographic studies indicated that the choroid plexus was the prime target for Cd uptake in the CNS for all age groups, with the blood vessels a target in the early postnatal period.

The effects of acute or chronic cadmium exposure on the adult mouse CNS: a freeze-fracture study.

The effect of acute Cd exposure (0.5-12 mg CdCl2/kg body wt. for six hours) or chronic Cd exposure (40 ppm in the drinking water for 3 weeks) on the CNS of adult mice was investigated quantitatively by the use of the freeze-fracture technique to examine the nuclear membrane of cells from the cerebral cortex. Intramembrane particle density and nuclear pore density was determined for both the P face of the inner membrane and the E face of the outer membrane from control and experimental animals. The changes observed for both groups of experimental animals were similar in that particle numbers for both P and E faces were significantly increased over control values while nuclear pore numbers were unaffected. For the acute doses the increases were dose-dependent.

In vitro assessment of individual and interactive effects of aromatic hydrocarbons on embryonic development of the rat.

There have been reports of disruption of embryonic development following exposure of pregnant women to aromatic hydrocarbons. In the present study, the embryotoxicity of toluene, xylene, benzene, styrene, and its metabolite, styrene oxide, was evaluated using the in vitro culture of postimplantation rat embryos. Possible interactions between toluene, xylene, and benzene were also studied using mixtures of these solvents. The results of the study showed that toluene, xylene, benzene, and styrene all have a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. Styrene was embryotoxic at a lower concentration (1.00 mumol/mL) than benzene (1.56 mumol/mL), toluene (2.25 mumol/mL), or xylene (1.89 mumol/mL). The metabolite of styrene, styrene oxide, was embryotoxic at a concentration (0.038 mumol/mL). more than 20 times less than the parent compound. There was no evidence of a synergistic interaction between toluene, xylene, and benzene in causing embryotoxicity; the solvents interacted in an additive manner. The embryos were exposed to the solvents for 40 h of the organogenic period. When the levels of solvents found to be embryotoxic in the present study are compared to blood levels in the human following industrial exposure or solvent abuse, it appears unlikely that the threshold blood levels for embryotoxicity would be exceeded in the workplace. However, the possibility that exposure to solvents earlier or later or throughout the entire organogenic period might result in a different conclusion cannot be excluded.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

The contribution of primary and secondary neuronal degeneration to prenatally-induced micrencephaly.

Prenatal exposure of rats to the alkylating agent methylazoxymethanol acetate (MAM Ac) induces severe micrencephaly in the offspring. The aim of the present study was to examine the contribution of primary cell death (due to a direct action of MAM Ac on the neuroepithelium), and secondary (target-dependent) cell death to the subsequent cell deficits in the visual system following prenatal exposure to MAM Ac on embryonic day 11, 12, 13, 14, 15 or 16. The results showed that when primary cell death substantially reduced the neuronal population of a target structure then there was increased target-dependent cell death in the neurons which normally project to that target. This was particularly evident in the dorsal lateral geniculate nucleus following exposure to MAM Ac on E15. Although the MAM Ac caused virtually no primary cell death in the embryonic precursor cells of the dLGN, the nucleus in the adult offspring was reduced by 87% compared with controls. This reduction was shown to be due to increased postnatal target-dependent, or secondary, cell death due to a severe reduction in layers III and IV of the occipital cortex. The cortical damage was due to primary cell death. Hence, primary cell death only partly accounts for the degree of micrencephaly seen in the offspring, consideration of secondary cell death is necessary to understand the total deficit.

Fetal brain damage in the rat following prenatal exposure to cocaine.

The aim of this study was to identify fetal brain damage induced by 1) prenatal cocaine exposure or 2) physical procedures causing temporary constriction or occlusion of the uterine vessels in pregnant rats. Brains were examined from rat fetuses killed 48 hours after the dam was given one or more intraperitoneal doses of cocaine (50-70 mg/kg) on day 16 of gestation. Only brains from fetuses with hemorrhage in the extremities were examined, as this indicated they had undergone a circulatory disturbance. Four of the 10 brains examined showed bilateral necrosis and cavitation in the cerebral cortex. There were also hemorrhage and ectopic outgrowths in the corpus striatum, bilateral cavitation in the brainstem and vacuolization in the lens of the eye. A similar type and distribution of damage was seen in rat fetal brains from dams treated by temporary occlusion of the uterine vessels or direct handling of the pregnant uterus on day 16 of gestation and examined 48 hours later. It is proposed that the procedures act through the common mechanism of constriction/occlusion of the uterine vessels. The damage to the fetuses appears to be due to hemorrhage from the fetal vessels and ischemia. These findings are discussed in relation to cocaine use during human pregnancy.

Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K(1).

It has been suggested that vitamin K(1) (phylloquinone) can cause genetic damage in rapidly dividing cells and that this should be considered in the risk/benefit analysis of the prophylactic use of vitamin K(1) in the newborn. Usual intramuscular administration of 1mg of vitamin K(1) to the newborn gives peak plasma levels of 1-2 microg/ml (approximately 2-4 microM). To investigate the possible harmful effects of high concentrations of vitamin K(1), rat embryos undergoing rapid cell division in the organogenic period were cultured for 46 hours in rat sera containing either 1, 10 or 100 microg of added vitamin K(1) per ml (2, 22 or 222 microM). At the end of the culture period the embryos were dissociated and the cells examined for evidence of DNA damage using the alkaline version of the comet assay. Control embryos were cultured in sera without added vitamin K(1) and positive controls were control embryos exposed to hydrogen peroxide at the end of the culture period. The results did not show any evidence of DNA damage in the vitamin K(1) exposed embryos. The positive controls showed a significant increase in tail length, moment and inertia. In conclusion, under the experimental conditions used, high concentrations of vitamin K(1) did not induce primary DNA damage in cells from rat embryos grown in vitro.

Effects of a desealant formulation, SR-51(®) and its individual components on the oxidative functions of mitochondria.

The Royal Australian Air Force has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977-mid-1990s was the cause of health problems. Particular concern was directed at a desealant chemical mixture known as SR-51(®). The current study, using in vitro submitochondrial assays, was designed to investigate the relative toxicities of the four components of SR-51(®) (Aromatic 150 solvent (Aro150), dimethylacetamide (DMA), thiophenol (TP) and triethylphosphate (TEP)). Based on the EC(50) values, TP and Aro150 were the most toxic components and were markedly more toxic than TEP and DMA.